Triphosgene-mediated couplings in the solid phase: Total synthesis of brachystemin A

Article abstract of DOI:10.1002/ejoc.201403145

A reproducible, highly efficient, and racemization-free protocol for the triphosgene-mediated solid-phase coupling of proteinogenic amino acids was developed. The efficiency of the reaction was demonstrated by the first total synthesis of the bioactive natural octacyclopeptide brachystemin A. This protocol will broaden the application of triphosgene in the synthesis of a wide range of peptides, including aromatic amino acids, N-alkylated amino acids, as well as proteinogenic amino acids.

Full text of DOI:10.1002/ejoc.201403145

SHORT COMMUNICATION
DOI: 10.1002/ejoc.201403145
Triphosgene-Mediated Couplings in the Solid Phase: Total Synthesis of
Brachystemin A
Lijing Fang,[a] Chunlei Wu,[a] Zaidan Yu,[a] Peng Shang,[a] Yongxian Cheng,[b]
Yuefeng Peng,*[a] and Wu Su*[a]
Keywords: Synthetic methods / Solid-phase synthesis / Amidation / Peptides / Total synthesis
A reproducible, highly efficient, and racemization-free proto- tocol will broaden the application of triphosgene in the syn-
col for the triphosgene-mediated solid-phase coupling of pro-
teinogenic amino acids was developed. The efficiency of the
reaction was demonstrated by the first total synthesis of the
bioactive natural octacyclopeptide brachystemin A. This pro-
thesis of a wide range of peptides, including aromatic amino
acids, N-alkylated amino acids, as well as proteinogenic
amino acids.
ides are highly electrophilic, which allows rapid amidation
of sterically and electronically less-reactive amines. In 2002,
Jung et al. reported an fluorenylmethyloxycarbonyl
(Fmoc)-based synthesis of cyclic peptides containing steri-
cally hindered secondary amines on a solid support in
which BTC was used as the coupling agent.[3] A highly ef-
ficient strategy was developed for the synthesis of DNA-
binding hairpin polyamides by using BTC-mediated cou-
pling of tert-butoxycarbonyl (Boc) protected aromatic
amino acids.[4] BTC has also shown application in the syn-
thesis of aromatic oligoamides by using automated peptide
synthesizers.[5] Although far superior to benzotriazole-
based protocols for couplings of N-methylated or aromatic
amino acids in both Fmoc-based and Boc-based SPPS,
Jung et al. found in the total synthesis of cyclosporine O
and omphalotin A that for unmethylated amino acids such
as Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Ile-OH, and
Fmoc-Trp-OH, the BTC-mediated coupling failed to drive
the reaction to completion. In these cases, the coupling re-
action had to be done by additional coupling cycles with
the use of HATU or HOAt/DIC as the reagents with ex-
tended reaction times (16 h).[3] It was assumed that pro-
teinogenic amino acids bearing an NH proton of the ur-
ethane function were susceptible to side reactions during
strong activation with the BTC reagent.[3] Owing to the low
efficiency of BTC-mediated proteinogenic amino acid cou-
plings, the BTC strategy has not been generally used in the
synthesis of peptides composed of proteinogenic amino
acids since the first report of this method by Falb et al. in
1999.[2b]
Introduction
The activation of amino acids for the formation of amide
bonds is a key step in solid-phase peptide synthesis (SPPS).
Following the growing interest in peptides, numerous cou-
pling reagents have been developed and have become com-
mercially available, mainly including carbodiimides {N,NЈ-
dicyclohexylcarbodiimide (DCC), N,NЈ-diisopropylcarbo-
diimide (DIC), 1-ethyl-3-[3-(dimethylamino)propyl]carbo-
diimide hydrochloride (EDC·HCl)} alone or plus additives
[hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotri-
azole (HOAt)], phosphonium salts [(benzotriazol-1-yloxy)-
tris(dimethylamino)phosphonium
hexafluorophosphate
(BOP), (benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate (PyBOP), bis(2-oxo-3-oxazolidinyl)-
phosphinic chloride (BOP-Cl), pentafluorophenyl diphenyl
phosphate (FDP), pentafluorophenyl diphenylphosphinate
(FDPP), etc.], and uronium (iminium) salts [O-(7-aza-1H-
benzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium
fluorophosphate (HBTU), O-(benzotriazol-1-yl)-N,N,NЈ,NЈ-
tetramethyluronium tetrafluoroborate (TBTU), O-(7-aza-
hexa-
benzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium
hexa-
fluorophosphate (HATU), etc.].[1] Triphosgene (BTC) as a
reagent putatively forms acid chlorides in situ;[2] acid chlor-
[a] Guangdong Key Laboratory of Nanomedicine, Institute of
Biomedicine and Biotechnology, Shenzhen Institutes of
Advanced Technology, Chinese Academy of Sciences
1068 Xueyuan Avenue, Shenzhen, Guangdong 518055, China
E-mail: yuefeng.peng@fda.hhs.gov
wu.su@siat.ac.cn
http://www.siat.ac.cn/
[b] State Key Laboratory of Phytochemistry and Plant Resources
in West China, Kunming Institute of Botany, Chinese Academy
of Sciences,
The growing need for new and more complex peptide
structures, particularly for biomedical studies and for the
large-scale production of peptides as drugs, demands the
manufacture of peptide products by efficient synthetic stra-
tegies, at reasonably low prices. It is highly desirable if one
132# Lanhei Road, Heilongtan, Kunming, Yunnan 650204,
P. R. China
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201403145.
7572
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Total Synthesis of Brachystemin A
versatile coupling reagent could be applicable for the cou- tive side reaction during BTC activation, we surmised that
pling of both proteinogenic amino acids and non-pro- increasing the ratio of BTC might produce Fmoc-Val-OMe
teinogenic amino acids including N-alkylated amino acids with better conversion. As a matter of fact, the activation
and aromatic amino acids. For this purpose, the use of BTC efficiency was significantly improved if 0.41 equiv. of BTC
in the activation of amino acids is quite attractive, because was used (Figure 1, trace b) without any new byproduct de-
BTC is inexpensive, unlike many condensation agents, and tected. Possibly because of the moisture in the reaction sys-
has proved to be very efficient in the synthesis of difficult tem, a small amount of Fmoc-Val-OH was left unreacted,
sequences such as N-methylated peptides and aromatic which could not be avoided by a further increase in the
polyamides. In view of the limitations in activating pro- BTC amount to 0.50 equiv. (Figure 1, trace c). Therefore,
teinogenic amino acids, the development of an efficient we believe that 0.41 to 0.50 equiv. of BTC is appropriate to
activation method for this type of amino acids is crucial activate proteinogenic amino acids efficiently and will lead
for the generality of the BTC strategy. Recently, Fuse and to a successful coupling in the SPPS.
Takahashi reported efficient amide bond formation through
rapid and strong activation of various carboxylic acids with
BTC in a microflow reactor.[6] Side reactions including epi-
merization were suppressed by reducing the residence time
of the highly active species generated through microflow
synthesis. Although this method performs well in solution
phase, it is not applicable for solid-phase synthesis, which
needs a longer time to accomplish the coupling. Therefore,
along with our continuous interest in the search for efficient
strategies in SPPS, we examined the problematic activation
of proteinogenic amino acids and describe herein a highly
efficient procedure for the BTC-mediated solid-phase syn-
thesis of proteinogenic peptides (Scheme 1).
Figure 1. Activation of Fmoc-Val-OH by using BTC.
Noting that the collidine hydrochloride salt precipitated
during BTC activation, which could be a major hindrance
to the application of this method to automated peptide syn-
thesis, we then set out to deal with the precipitate and fi-
nally developed a novel procedure by using a special combi-
nation of bases for the activation of the amino acids. The
key features of the procedure include: (1) performing the
preactivation of the carboxylic acid with collidine in THF,
racemization-free conditions validated by the research of
Jung and Falb; (2) conversion of the resulting precipitate
into the soluble N,N-diisopropylethylamine (DIEA) hydro-
chloride salt by the addition of a DIEA/DMF solution. For
a typical coupling reaction on 150 mg resin (0.36 mmolg–1),
Scheme 1. BTC-mediated solid-phase coupling of proteinogenic
amino acids.
Results and Discussion
We first chose Fmoc-Val-OH to evaluate the activation the Fmoc-protected amino acid (4 equiv.) and BTC
efficiency of the BTC method. A series of experiments was (1.64 equiv.) were dissolved in dry THF (1 mL); then collid-
performed by using different ratios of BTC and Fmoc-Val- ine (14 equiv.) was added slowly and a white solid precipi-
OH. According to Jung’s procedure, Fmoc-Val-OH and tated immediately. This suspension was gently shaken for
BTC were dissolved in dry THF, collidine was added slowly, about 3 min before DIEA/DMF (5% v/v, 2 mL) was added
upon which a white precipitate formed. This suspension to form a clear solution, which was transferred to the pre-
was gently shaken for about 2 min before an excess amount treated resin to generate the corresponding peptide.
of dry MeOH was added to generate Fmoc-Val-OMe to
Encouraged by these results, we investigated the effi-
facilitate the HPLC analysis. As shown in Figure 1, we ciency of the optimized BTC protocol to form amide bonds
found that 0.33 equiv. of BTC, typically used in the acti- in solid-phase peptide synthesis. For a model study, Fmoc-
vation step to release an equal molar amount of phosgene, Ala-OH, Fmoc-Val-OH, and Fmoc-Ile-OH were chosen to
was not sufficient to completely convert the amino acid into prepare a short peptide on Wang resin preloaded with
the corresponding methyl ester (Figure 1, trace a). Con- Fmoc-Gly. As reported in the total synthesis of cyclospor-
sidering the NH proton of the Fmoc-protected amino ine O and omphalotin A, the previous BTC procedure was
group might consume some phosgene through a competi- useless for the coupling of these amino acids. To our de-
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L. Fang, C. Wu, Z. Yu, P. Shang, Y. Cheng, Y. Peng, W. Su
SHORT COMMUNICATION
light, the solid-phase synthesis of resin-bound Gly-Ala-Val- brachystemin A, originally proposed as cyclo-(Pro1-Phe2-
Ile-Fmoc proceeded smoothly by using the optimized BTC Leu3-Ala4-Thr5-Pro6-Ala7-Gly8), was revised to cyclo-
protocol. Cleavage of the tetrapeptide from the resin af- (Pro1-Pro2-Ala3-Gly4-Leu5-Ala6-Thr7-Phe8) as a result of
forded the crude product in 86% purity; this is indicative QTOF/MS and X-ray diffraction analysis (Figure 4).[8] It
of an average yield of 95% per coupling cycle (Figure 2).
was shown that bachystemin A, at a concentration of 10 μm,
could significantly inhibit the secretion of IL-6, CCL-2, and
collagen IV. In addition, its effects on collagen I, reactive
oxygen species (ROS), production of the superoxide anion
(O2·–), cell viability in mesangial cells, and nitric oxide pro-
duction in macrophage cells were evaluated, and it was re-
vealed that brachystemin A was a promising agent for the
treatment of diabetic nephropathy (DN).[9] Further explora-
tion of this octacyclopeptide in drug discovery was, how-
ever, prevented by the lack of its availability from natural
sources. Therefore, we initiated the total synthesis of bachy-
stemin A by head-to-tail cyclization of a linear peptide.
Furthermore, this octacyclopeptide not only contains the
glycine and alanine residues used in our model study, but it
also contains special residues such as Phe, Thr, and Pro; it
therefore appeared as an ideal target on which to test the
efficiency of our newly developed BTC protocol.
Figure 2. HPLC of crude linear tetrapeptide.
Another model reaction was used to check for racemiza-
tion of the optimized BTC protocol. Coupling of racemic
Fmoc-Ala-OH and Fmoc-l-Ala-OH or Fmoc-d-Ala-OH to
resin-bound Gly-Val-NH2 was performed, and the resulting
tripeptide was subjected to HPLC (Figure 3). For the cases
in which enantiomerically pure Fmoc-d-Ala-OH and
Fmoc-l-Ala-OH were used, no racemization products
could be detected.
Figure 4. Brachystemin A (1).
It was envisioned that the existence of two proline resi-
dues, which can induce conformational turns, should en-
courage head-to-tail peptide cyclization. With circumspec-
tion, a cyclization point at the Gly4-Leu5 junction was cho-
sen to give the linear precursor NH2-Leu-Ala-Thr-Phe-Pro-
Pro-Ala-Gly-OH (2). This precursor was expected to cyclize
readily while failing to support any C-terminal epimeri-
zation during the cyclization.
Linear octapeptide 2 was prepared by using Fmoc-based
SPPS techniques with Fmoc-Gly-Wang resin as the solid
support and BTC as the coupling reagent (Scheme 2). As
described in the model study, the activation of Fmoc-Xaa-
OH was completed with BTC (0.41 equiv.) in 5 min; cou-
pling times of 30 min to 1 h were typically required for
quantitative conversion to the coupled products according
to the ninhydrin test (for the free primary amine) and the
Figure 3. Comparison of the HPLC traces of tripetides resulting
from BTC-mediated coupling of (a) racemic Fmoc-Ala-OH,
(b) Fmoc-l-Ala-OH, and (c) Fmoc-d-Ala-OH (CHIRALPAK ID
0.46 cmϫ25 cmϫ5 μm, Shimadzu LC 20 with UV detector SPD-
20A, 254 nm).
We then investigated whether the BTC-based coupling chloranil test (for the free secondary amine of Pro). Depro-
methodology could be applied to the preparation of biolo- tection of the Fmoc group was performed with 20% piper-
gically active cyclopeptides. Brachystemin A (1) is an octa- idine in DMF. Cleavage of the precursor from the resin was
cyclopeptide that was isolated from Brachystemma calyci- achieved with trifluoroacetic acid (TFA)/triethylsilane
num D. Don (Caryophyllaceae), a Chinese folk medicine (TES)/H2O (95:2.5:2.5).[10] Owing to the high purity, the
that has been used to treat rheumatoid arthritis, limb numb- linear peptide could be cyclized after precipitation from
ness, impotence, and gonorrhea.[7] In 2011, the structure of cold diethyl ether without further purification.[11]
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Eur. J. Org. Chem. 2014, 7572–7576

Total Synthesis of Brachystemin A
amino acids, which resulted in the development of a repro-
ducible, low-costing, and very efficient protocol for BTC-
mediated solid-phase peptide synthesis. The optimized BTC
method was successfully applied to the first total synthesis
of brachystemin A, a naturally occurring, bioactive cyclic
octapeptide. This precedent will broaden the application of
BTC in the synthesis of a wide range of peptides including
aromatic amino acids, N-alkylated amino acid, as well as
proteinogenic amino acids. It also shows potential for auto-
mation in multiple, parallel peptide synthesis, which we are
currently investigating in our laboratory. We anticipate that
the automated synthesis of brachystemin A will provide
sufficient material for further pharmacokinetics and phar-
macodynamics studies in animals.
Scheme 2. Total synthesis of brachystemin A.
Experimental Section
General: All reagents were HPLC or peptide-synthesis grade. Dry
DMF and dry THF were obtained from Sigma–Aldrich. Collidine
(2,4,6-collidine) was purchased from J&K Scientific. BTC, DIEA,
and TFA were purchased from Aladdin. Fmoc-Gly-Wang resin and
the amino acids were purchased from GL Biochem. All commercial
reagents were used as received. Analytical reverse-phase HPLC was
performed at room temperature with a Shimadzu LC 20 with a UV
detector SPD-20A by using an Inertsil ODS-SP column
(4.6ϫ250 mm, 5 μm, 100 Å). The reverse-phase HPLC gradient
was started at 10% of B (MeCN), then increased to 100% of B
over 30 min (A: 0.1% TFA in water). 1H NMR (13C NMR) spectra
were recorded with a Bruker Avance AV500, Bruker Avance AV400,
or DPX-300 at 500 (125), 400 (100), or 300 MHz (75 MHz), respec-
tively. Chemical shifts are referenced to either tetramethylsilane as
an internal standard or the signals resulting from the residual sol-
vent. Mass spectra were measured with an ABI Q-star Elite.
In the following study, the coupling reagents that could
affect the head-to-tail cyclization of linear precursor 2 were
screened. Ring closure was effected by EDC·HCl, HATU,
HBTU, or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-
–
morpholinium tetrafluoroborate (DMTMM+BF4 ) at high
dilution (5 mm) in the various yields presented in Table 1.
As expected, octacyclopeptide
1
was isolated in
every case in moderate to good yield. Recently,
DMTMM+BF4– was found to be effective in fragment cou-
pling and cyclization to provide high yields and a low de-
gree of epimerization for a wide range of amino acid and
peptide substrates.[12] Cyclization of 2 by using this cou-
pling reagent gave the highest yield of product (72%). Use
of the much cheaper chloride salt, DMTMM+Cl–, provided
1 in 59% yield, which was lower than that obtained with
HATU/DIEA as the coupling reagents but higher than that
obtained with EDCI/HOBt or HBTU/DIEA as the cou-
pling reagents. Purification of the crude product by prepar-
ative reverse-phase HPLC followed by lyophilization af-
BTC Activation of Fmoc-Val-OH: Fmoc-Val-OH (1.0 equiv.) and
BTC (0.41 equiv. or test equiv.) were dissolved in dry THF (1 mL).
Collidine (14 equiv.) was added slowly to the resulting solution, and
the suspension was gently shaken for about 2 min before an excess
amount of dry MeOH (1.0 mL) was added. The mixture was stirred
at room temperature for 2 h before a small portion was taken out
for HPLC analysis. CAUTION: Phosgene and CO2 gas were
evolved; this operation must be performed in a fume hood!
1
forded 1 as a white powder. The H NMR and 13C NMR
(500 and 75 MHz) spectra of 1 and the mass of the HRMS
parent ion were identical to those of natural brachyste-
min A (see the Supporting Information).
Synthesis of Linear Octapeptide 2: Polyamide synthesis was per-
formed manually in a 10 mL peptide synthesis vessel by solid-phase
Fmoc chemistry. Fmoc-Gly-Wang resin was used as a solid support
at a substitution level of 0.36 mmolg–1 (150 mg, 0.054 mmol). The
Fmoc group was deprotected by using 20% piperidine/DMF. After
washing with DMF (4ϫ 2 mL), the resin was treated with dry
DMF (2 mL) for 2 min. Meanwhile, the following Fmoc-protected
amino acid (4.0 equiv.) and BTC (1.64 equiv.) were dissolved in dry
THF (1 mL), and the resulting solution was cooled to 0 °C in an
ice/water bath. Collidine (14 equiv.) was added slowly to the THF
solution, and a white solid precipitated immediately. After activa-
ting for 3 min, DIEA/DMF (5% v/v, 2 mL) was added to form a
clear solution, which was transferred to the deprotected Wang
resin. The mixture was shaken for 0.5 to 1 h (until the Kaiser test
was negative for the free primary amine or the chloranil test was
negative for the free secondary amine of proline), and it was then
drained from the resin and rinsed with DMF (4ϫ 2 mL). This pro-
cedure was repeated until a peptide sequence (NH2-Leu-Ala-Thr-
Phe-Pro-Pro-Ala-Gly) bound to Wang resin was obtained. After
Table 1. Yields of isolated 1 formed by cyclization of 2 by using
different coupling reagents.
Coupling reagents
Yield [%][a]
EDC·HCl/HOBt
HBTU/DIEA
HATU/DIEA
53
53
68
72
59
DMTMM+BF4 /DIEA
–
DMTMM+Cl–/DIEA
[a] The cyclizations were performed in DMF at high dilution
(5 mm) of linear peptide 2.
Conclusions
We demonstrated that increasing the molar ratio of tri-
phosgene (BTC) to the amino acids significantly improved
the activation efficiency of unmethylated proteinogenic washing with DMF (4ϫ 2 mL) and MeOH (4ϫ 2 mL), the resin
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7575

L. Fang, C. Wu, Z. Yu, P. Shang, Y. Cheng, Y. Peng, W. Su
SHORT COMMUNICATION
was then dried under N2. Peptide 2 was cleaved off the Wang resin
by using TFA/TES/H2O (95:2.5:2.5, 1.0 mL) at room temperature
for 4 h. The TFA/Wang resin/peptide mixture was then filtered to
remove the resin. The crude peptide was precipitated by adding
eight volumes of diethyl ether and cooling to 0 °C. The crude prod-
uct was collected by centrifugation and dried under vacuum to pro-
duce a white powdery solid, which was used for cyclization without
further purification (39 mg). HRMS (ESI): calcd. for C37H57N8O10
[M + H]+ 773.4198; found 773.4196.
ful for the assistance of the NMR and mass facility from Peking
University, Shenzhen Graduate School. We are grateful to Dr.
G. W. A. Milne for manuscript revision.
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Typical Procedure for the Synthesis of Cyclopeptide 1: DIEA
–
(19 μL, 0.11 mmol) and DMTMM+BF4 (33 mg, 0.1 mmol) were
added sequentially to
a solution of octapeptide 2 (39 mg,
0.05 mmol) in DMF (10 mL). The resulting solution was stirred at
room temperature for 72 h, and then the solvent was removed un-
der vacuum, and the residue was precipitated with water. The crude
product was collected by centrifugation and purified by semipre-
parative reverse-phase HPLC followed by lyophilization to afford
1
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1 as a white powder (27 mg, 72%). Data for synthetic 1: H NMR
3910–3913.
(500 MHz, C5D5N): δ = 10.56 (s, 1 H), 8.94 (t, J = 6.0 Hz, 1 H),
7.94 (d, J = 9.5 Hz, 1 H), 7.70 (d, J = 5.5 Hz, 1 H), 7.50 (d, J =
7.5 Hz, 2 H), 7.32–7.28 (m, 2 H), 7.28–7.14 (m, 3 H), 5.38 (t, J =
4.5 Hz, 1 H), 5.22–5.15 (m, 1 H), 5.05–4.95 (m, 3 H), 4.79 (d, J =
8.5 Hz, 1 H), 4.62–4.54 (m, 2 H), 4.31–4.29 (m, 1 H), 3.98 (t, J =
9.0 Hz, 1 H), 3.83 (dd, J = 17.0, 6.0 Hz, 1 H), 3.45–3.38 (m, 3 H),
3.30 (t, J = 12.0 Hz, 1 H), 3.13 (t, J = 8.0 Hz, 1 H), 2.31–2.27 (m,
2 H), 2.18–2.10 (m, 1 H), 2.08–1.96 (m, 3 H), 1.93–1.84 (m, 1 H),
1.84 (d, J = 7.5 Hz, 3 H), 1.80–1.75 (m, 1 H), 1.56 (d, J = 7.0 Hz,
3 H), 1.62–1.45 (m, 2 H), 1.39 (d, J = 6.0 Hz, 3 H), 1.33–1.23 (m,
1 H), 0.96 (d, J = 6.5 Hz, 3 H), 0.74 (d, J = 6.0 Hz, 3 H) ppm. 13C
NMR (75 MHz, C5D5N): δ = 177.2, 173.9, 173.7, 172.3, 172.1,
171.9, 169.7, 169.5, 138.8, 130.0, 128.9, 127.2, 66.5, 64.6, 64.0, 59.7,
55.7, 53.5, 50.1, 48.7, 47.5, 47.2, 44.2, 44.0, 36.5, 29.1, 28.2, 26.4,
25.1, 24.7, 23.8, 21.9, 21.1, 18.8, 16.8 ppm. HRMS (ESI): calcd. for
C37H54N8O9Na [M + Na]+ 777.3912; found 777.3906.
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[9] a) J. F. Navarro, C. J. Mora, J. Am. Soc. Nephrol. 2008, 19,
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[10] Notably, the hydroxy group of threonine was protected as a
tert-butyl ether during the linear peptide Synthesis Deprotec-
tion took place simultaneous to cleavage of octapeptide 2 from
the resin by using a mixture of TFA/TES/H2O.
Supporting Information (see footnote on the first page of this arti-
cle): Copies of the 1H NMR and HR mass spectra of all key inter-
mediates and final products.
[11] A major and minor set of resonances in a 2:1 ratio is clearly
observed in the 1H NMR spectrum (D2O), which indicates that
linear peptide 2 exists as rotational conformers.
Acknowledgments
[12] a) Z. J. Kaminski, B. Kolesinska, J. Kolesinska, G. Sabatino,
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Received: August 29, 2014
This work was supported by the “Hundred Talents Program”
of the Chinese Academy of Sciences, the Shenzhen Sciences &
Technology Innovation Council (CXZZ20120831175831692,
KQCX20130628112914285) and the National Natural Science
Foundation of China (Grant No. 21402232). The authors are grate-
Published Online: October 21, 2014
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